The CRISPR family enzyme Cas13 at work. Cas13 (pink), is at the heart of the RESCUE platform, where it uses a special guide (red) to target RNAs in the cell (blue). [Stephen Dixon]

CRISPR-based gene editing tools have revolutionized how scientists can target gene mutations and manipulate gene expression. Such tools hinge on the use of the enzymes Cas9 and Cas12 to target DNA, and Cas13 to target RNA.

Modifying RNA can have some advantages as it avoids permanent modifications to the genome, and could make CRISPR technology more applicable to some cell types, such as neurons, which have proven hard to edit using CRISPR-Cas9-based approaches. Researchers headed by Feng Zhang, PhD, a McGovern Institute researcher and core member of the Broad Institute of MIT and Harvard, now report on a new approach that combines a deactivated Cas13 with a novel, programmable enzyme to target and then convert specific cytosine bases to urindine (C-to-U conversion) on targeted RNA transcripts.

The technique, which has the acronym RESCUE (RNA editing for specific C to U exchange), builds on the team’s existing REPAIR (RNA editing for programmable A to I (G) replacement) technology, which converts Cas13-targeted adenine residues to inosine. Reporting in Science the researchers describe initial experiments through which RESCUE was used to transiently and reversibly increase β-catenin activation and cell growth, and to convert the APOE4 variant into APOE2. They suggest that the new technology significantly expands the scope of CRISPR-based tools, making it possible to modify key positions on proteins, such as phosphorylation sites, for the first time. Protein phosphorylation sites can act as an on/off switches for protein activity and are found in signaling molecules and cancer-linked pathways.

“To treat the diversity of genetic changes that cause disease, we need an array of precise technologies to choose from,” Zhang commented. “By developing this new enzyme and combining it with the programmability and precision of CRISPR, we were able to fill a critical gap in the toolbox.” Zhang is the James and Patricia Poitras professor of neuroscience at MIT. Zhang also has appointments in MIT’s departments of brain and cognitive sciences and biological engineering. The researchers’ published paper describing the RESCUE technology is titled “A cytosine deaminase for programmable single-base RNA editing.”

Zhang’s previously developed REPAIR technology uses Cas13 to direct the catalytic domain of an RNA editing enzyme, adenosine deaminase acting on RNA 2 (ADAR2) to target sites on RNA, where it converts adenosine to inosine, with single base precision. However, the authors pointed out, REPAIR and other RNA editing technologies only allow for A-to-I conversions. “Technologies for precise RNA editing of cytidine to uridine would greatly expand the range of addressable disease mutations and protein modifications.”

Natural enzymes that can catalyze C-to-U conversion have been used for DNA base editing, but they only operate on single-stranded nucleic acids, and may exhibit off-target editing. To get around these problems the team built their own system, by evolving the adenine deaminase domain of ADAR2, which naturally acts on double-stranded RNA substrates, into a cytidine deaminase. The resulting evolved ADAR2 was then fused to a deactivated Cas13 (dCas13), and further evolved into the RESCUE tool.

The team tested the new platform in cultured human cells, demonstrating that they could precisely target natural RNAs in cells, and also target 24 clinically relevant mutations in synthetic RNAs. They then further optimized RESCUE to reduce off-target editing, but without significantly disrupting on-target editing.

A major advantage of RNA editing is that it is reversible, in contrast with DNA-level editing which is permanent, and so RESCUE can be used to to make transient, temporary modifications in RNA. Zhang’s team demonstrated this in human cells, by using RESCUE to target specific sites in the RNA encoding β-catenin that are known to be phosphorylated on the protein product. The modifications resulted in a temporary increase in β-catenin activation and cell growth. “We tested a panel of guides targeting the β-catenin transcript (CTNNB1) at known phosphorylation residues and observed editing levels between 5% and 28%, resulting in up to 5-fold activation of Wnt/β-catenin signaling and increased cell growth …” the team wrote. If such a change had been permanent it could have resulted in uncontrolled cell growth and cancer, whereas exploiting RESCUE to promote transient cell growth could potentially stimulate wound healing in response to acute injuries.

The researchers also used RESCUE to target ApoE4, a variant of the ApoE protein that is widely considered a genetic risk factor for late-onset Alzheimer’s disease. The ApoE4 isoform differs from the non-pathogenic ApoE2 by two single base changes in sequence, both of which are C in ApoE4, and U in ApoE2. Zhang and colleagues introduced the risk-associated ApoE4 RNA into cells, and demonstrated how RESCUE could convert the ApoE4-specific cytidines back to uridine, effectively restoring the ApoE2 sequence, and so converting the risk variant to a non-risk variant.

The ability for C-to-U conversion gives researchers the opportunity to use RESCUE to target sites that regulate protein activity through post-translational modifications such as phosphorylation, glycosylation, or methylation. “RESCUE doubles the number of pathogenic mutations targetable by RNA editing and enables modulation of phospho-signaling-relevant residues,” the authors stated. “The larger targetable amino acid codon space of RESCUE enables modulation of more post-translational modifications, such as phosphorylation, glycosylation, and methylation, as well as expanded targeting of common catalytic residues, disease mutations, and protective alleles, such as ApoE2.”

The Zhang lab aims to share the RESCUE system with the scientific community, to encourage additional research that could move RESCUE close to the clinic, as well as give researchers a new tool to better understand disease-causing mutations. The technology will be freely available for academic research through the non-profit plasmid repository Addgene.

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